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. 2012 Jan;10(1):e1001238.
doi: 10.1371/journal.pbio.1001238. Epub 2012 Jan 10.

USP8 promotes smoothened signaling by preventing its ubiquitination and changing its subcellular localization

Ruohan Xia  1 , Hongge Jia, Junkai Fan, Yajuan Liu, Jianhang Jia
Affiliations

USP8 promotes smoothened signaling by preventing its ubiquitination and changing its subcellular localization

Ruohan Xia et al. PLoS Biol. 2012 Jan.

Abstract

The seven transmembrane protein Smoothened (Smo) is a critical component of the Hedgehog (Hh) signaling pathway and is regulated by phosphorylation, dimerization, and cell-surface accumulation upon Hh stimulation. However, it is not clear how Hh regulates Smo accumulation on the cell surface or how Hh regulates the intracellular trafficking of Smo. In addition, little is known about whether ubiquitination is involved in Smo regulation. In this study, we demonstrate that Smo is multi-monoubiquitinated and that Smo ubiquitination is inhibited by Hh and by phosphorylation. Using an in vivo RNAi screen, we identified ubiquitin-specific protease 8 (USP8) as a deubiquitinase that down-regulates Smo ubiquitination. Inactivation of USP8 increases Smo ubiquitination and attenuates Hh-induced Smo accumulation, leading to decreased Hh signaling activity. Moreover, overexpression of USP8 prevents Smo ubiquitination and elevates Smo accumulation, leading to increased Hh signaling activity. Mechanistically, we show that Hh promotes the interaction of USP8 with Smo aa625-753, which covers the three PKA and CK1 phosphorylation clusters. Finally, USP8 promotes the accumulation of Smo at the cell surface and prevents localization to the early endosomes, presumably by deubiquitinating Smo. Our studies identify USP8 as a positive regulator in Hh signaling by down-regulating Smo ubiquitination and thereby mediating Smo intracellular trafficking.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Multi-monoubiquitination of Smo in Drosophila S2 cells.
(A) S2 cells were transfected with Myc-Smo alone or in combination with HA-Ub, followed by immunoprecipitation and Western blot analysis with the indicated antibodies. A blank vector was used as the immunoprecipitation control, GFP as the transfection control, and IgG as the loading control. (B) Myc-Smo was transfected in S2 cells with or without HA-Ub followed by immunoprecipitation and Western blot with the indicated antibodies. The expression of HA-Ub did not cause any changes in the levels of Smo ubiquitination. The levels of HA-Ub are comparable to the levels of free Ub detected by P4D1 (bottom panel). (C) Myc-Smo was co-transfected with HA-Ub or with each individual HA-tagged Ub mutant, followed by immunoprecipitation and Western blot analysis. HA-Ub and Ub mutants gave rise to a similar smear pattern (top panel).
Figure 2
Figure 2. Addition of Ub to the C-terminus of Smo attenuates the signaling activity of Smo.
(A) A wild-type adult wing indicates the five interveins. (B–D) Wings expressing Myc-Smo, Myc-SmoUb, or Myc-SmoCFP at the 75B1 attP locus by MS1096 Gal4 to ensure equal expression of each individual constructs. The arrows in (B) and (D) indicate the extra wing structures in the anterior compartment of the wing, which is indicative of anterior ectopic Hh activity that is caused by the expression of wild-type Myc-Smo or Myc-SmoCFP. The wing in (C) shows the wild-type structure, which indicates that the activity of Myc-SmoUb is lower than that of Myc-Smo or Myc-SmoCFP. (E–F) S2 cells were transfected with RFP-Rab7 and GFP-Smo or GFP-SmoUb followed by immunostaining with Rab5 antibody to label the early endosomes. Late endosomes are labeled by RFP-Rab7. GFP indicates the expression and localization of Smo. The images shown in (E–F) are from intensity projection over the z-axis. (G) The percentage of Smo that co-localized with Rab5 or Rab7 compared to the total amount of Smo (GFP puncta) calculated from (E–F) (n≥10). More GFP-SmoUb puncta were localized in Rab7-labeled late endosomes compared to GFP-Smo. (H) S2 cells were transfected with Myc-Smo, Myc-SmoUb, or Myc-SmoCFP. Cell extracts were immunoprecipitated with the anti-Myc antibody and blotted with the anti-Myc antibody to examine the levels of Smo. GFP served as a transfection and loading control. Of note, the stability of Myc-SmoUb is much lower than that of Myc-Smo or Myc-SmoCFP. (I) S2 cells were transfected with Myc-Smo, Myc-SmoUb, or Myc-SmoCFP followed by treatment with or without Hh. Cell extracts were subjected to immunoprecipitation and Western blot with the anti-Myc antibody to detect the levels of Smo. Note that Hh stabilizes Myc-Smo (lane 2, top panel) but not Myc-SmoUb (lane 4, top panel). GFP served as a transfection and loading control.
Figure 3
Figure 3. Smo ubiquitination is regulated by Hh and phosphorylation.
(A) S2 cells were transfected with Myc-Smo and treated with 30% (lanes 2 and 5) or 60% (lanes 3 and 6) of Hh-conditioned medium or control medium (lanes 1 and 4), followed by treatment with or without NH4Cl. Cell extracts were immunoprecipitated and a Western blot was performed with the indicated antibodies. IgG served as a loading control and GFP was used as the transfection control. The arrow indicates hyperphosphorylated forms of Smo and the arrowhead indicates hypophosphorylated and unphosphorylated forms. (B) Sequential treatments with Hh and lysosomal inhibitor induce differential ubiquitination of Smo. S2 cells were transfected with Myc-Smo and treated with either Hh or the lysosomal inhibitor NH4Cl alone, or in combinations of Hh and NH4Cl treatment, followed by immunoprecipitation and Western blot with the indicated antibodies to examine the ubiquitination levels of Smo. The following treatments were performed: lane 1, control; lane 2, treatment with NH4Cl at a final concentration of 10 mM for 24 h; lane 3, treatment with 60% HhN-conditioned medium for 24 h; lane 4, treatment with NH4Cl at the concentration of 10 mM for the first 6 h, then adding 60% of the volume of HhN-conditioned medium, keeping NH4Cl at a final concentration of 10 mM for 18 h; lane 5, treatment with 60% volume of HhN-conditioned medium for the first 6 h, then adding NH4Cl at final concentration of 10 mM, keeping 60% volume of Hh medium for 18 h; lanes 6 and 7, HhN cDNA was cotransfected to achieve higher levels of Hh treatment. Consistently, Hh treatment down-regulates (lane 3, top panel) whereas NH4Cl treatment up-regulates (lane 2, top panel) Smo ubiquitination. However, Hh treatment down-regulates Smo ubiquitination before NH4Cl treatment (lane 5, top panel), but not after NH4Cl treatment (lane 4, top panel). The further down-regulation of Smo ubiquitination by Hh was achieved by cotransfecting Hh cDNA (lane 6, compared to lane 3). (C) NIH3T3 cells were transfected with Myc-hSmo and treated with or without recombinant Shh. Shh induced a marked decrease in hSmo ubiquitination (lane 3). (D) HEK293T cells were transfected with the indicated constructs and cell extracts were immunoprecipitated and blotted with the indicated antibodies. Cell lysates were also subjected to direct Western blot to examine the HA-tagged Ub expression. GFP served as transfection and loading control. Human Smo had a similar multi-mono ubiquitination pattern compared to fly Smo. (E) S2 cells transfected with Myc-Smo were treated with Hh-conditioned medium or control medium in combination with a PKA inhibitor (H-89) or CK1 inhibitor (CK1-7). Immunoprecipitation and Western blots were performed with the indicated antibodies. The levels of ubiquitinated Smo were increased by inactivating kinases. (F) S2 cells were transfected with wild-type, phospho-mimetic, or phospho-deficient Smo followed by treatment with Hh-conditioned medium or control medium. The same ubiquitination assay was carried out to examine ubiquitinated Smo with IgG and GFP serving as controls.
Figure 4
Figure 4. Identification of USP8 as a DUB for Smo.
(A) S2 cells were transfected with Myc-Smo and treated with the indicated DUB dsRNA, followed by immunoprecipitation with an anti-Myc antibody and Western blot with the P4D1 antibody to examine the levels of Smo ubiquitination. (B) Myc-Smo was transfected either alone or in combination with Flag-USP8, Flag-USP8C>S, or USP8 dsRNA treatment. The immunoprecipitation assay was performed to examine the amount of ubiquitinated Smo. The efficiency of USP8 RNAi was examined with the anti-USP8 antibody. The arrowhead indicates transfected USP8 and the arrow indicates endogenous USP8. Notably, USP8 RNAi and USP8C>S severely destabilized Smo, whereas USP8 stabilized Smo in S2 cells. (C) S2 cells were transfected with Myc-Smo and the indicated DUB constructs followed by immunoprecipitation with the anti-Myc antibody and Western blot with the anti-Ub P4D1 antibody. Flag-USP8, but not other DUBs, reduced Smo ubiquitination (lane 3, compared to lane 2, top panel). Cell lysates were also subjected to Western blot to examine the expression of different DUBs (bottom panel). (D) S2 cells were transfected with the indicated constructs and an immunoprecipitation assay was performed to detect Smo ubiquitination. The asterisks in (B) and (D) show a possible modification on USP8C>S, which was down-regulated by the co-transfection of USP8, suggesting the possibility of ubiquitination.
Figure 5
Figure 5. USP8 regulates Smo ubiquitination.
(A) S2 cells transfected with Myc-Smo in combination with the indicated USP8 constructs or with USP8 dsRNA treatment were treated with Hh-conditioned medium or control medium. An immunoprecipitation assay was carried out with the indicated antibodies to examine the regulation of Smo ubiquitination by Hh or USP8. In the above experiments, GFP served as a transfection and loading control. (B) S2 cells were transfected with Myc-SmoSD123 alone or in combination with either Flag-USP8, Flag-USP8C>S, or USP8 dsRNA treatment. Cell extracts were immunoprecipitated with the anti-Myc antibody and subjected to a Western blot with the anti-Ub P4D1 antibody to detect ubiquitinated Smo (top panel), or with the anti-Myc antibody to detect the expression of Myc-SmoSD123 (upper-middle panel). Cell lysates were also subjected to a direct Western blot with the anti-Flag antibody to detect USP8 or USP8C>S overexpression (lower-middle panel) or with the anti-Ub P4D1 antibody to detect the overall level of Ub (bottom panel). The asterisk indicates a possible modification of USP8C>S. USP8 reduced SmoSD123 ubiquitination, whereas USP8C>S enhanced SmoSD123 ubiquitination. The asterisks in (A) and (B) show a possible modification on USP8C>S. (C) The interaction between Smo fragments and USP8 was determined in S2 cells. Briefly, S2 cells were co-transfected with Flag-USP8 and each Myc-Smo construct followed by immunoprecipitation with the anti-Myc antibody and Western blot with anti-Flag antibody to detect the Smo-bound USP8. Smo1-555 (N-terminal part of Smo) did not interact with USP8 (not shown). The +/− indicates a weak interaction. (D) Hh up-regulates Smo-USP8 interaction. Extracts from S2 cells expressing Flag-USP8 with or without Hh treatment were incubated with the bacterially expressed GST or GST-Smo625-753 fusion proteins. The bound USP8 proteins were analyzed by Western blot with the anti-Flag antibody. The middle panel shows the GST and GST-Smo fusion proteins. The lower panel indicates the equal amount of input USP8. (E) The interaction between Flag-USP8 and GST-Smo625-753, GST-Smo625-753SD123 (GST-SmoSD, with PKA and CKI1 sites mutated to Asp), and GST-Smo625-753PKA123 (GST-SmoSA, with PKA sites mutated to Ala). (F) S2 cells were co-transfected with HA-USP8 and wild-type, phospho-mimetic, or phospho-deficient Smo, followed by immunoprecipitation and Western blot analysis to examine the interaction between Smo and USP8. GFP served as a transfection and loading control. (G) S2 cells were transfected with Myc-Smo alone or in combination with Flag-USP8 and treated with Hh-conditioned medium or control medium. Cell extracts were immunoprecipitated with the anti-Myc antibody and blotted with either anti-Myc to detect Smo phosphorylation, indicated by its mobility shift on the SDS gel, or anti-SmoP, to directly detect Smo phosphorylation. The overexpression of USP8 increased the level of Smo without any effect on Smo phosphorylation. GFP served as a transfection and loading control.
Figure 6
Figure 6. The function of USP8 in regulating Smo accumulation in the Drosophila wing.
(A–A″) A wing disc expressing UAS-GFP by the dorsal compartment-specific ap-Gal4 was stained for Smo and ptc-lacZ to show the wild-type staining. (B–B″) A wing disc from flies co-expressing UAS-USP8RNAi and UAS-GFP by ap-Gal4 was stained for Smo and ptc-lacZ. The arrow in (B) indicates the attenuated accumulation of Smo and the arrowheads indicate the down-regulation of ptc-lacZ. GFP indicates that ap-Gal4 is expressed in the dorsal compartment cells of the wing disc. (C–D) Wing discs expressing UAS-Flag-USP8C>S by MS1096 Gal4 were stained for Smo (grey in C), Flag (green in C′), and ptc-lacZ (red in D). The arrow in (C) indicates inhibition of Smo accumulation. The arrow in (D) indicates attenuated ptc-lacZ expression. Note that the level of Gal4 driven by MS1096 is higher in the dorsal compartment than in the ventral compartment. (E–E″) A wing disc bearing usp8KO homozygous clones, which were marked by the lack of β-gal staining, was immunostained for Smo. The arrow in (E) shows the reduced Smo accumulation in P-compartment cells. (F–G) Wing discs expressing UAS-Flag-USP8 by MS1096 Gal4 were stained for Smo (grey in F), Flag (green in F′), and ptc-lacZ (red in G). Arrow and arrowhead in (F) indicate the elevated Smo and arrow in (G) indicates the expansion of ptc-lacZ. (H) A wild-type embryo was stained for En. (I–J) Embryos expressing USP8RNAi or USP8C>S by act5C-Gal4 were immunostained for En. The arrowheads indicate the reduction and contraction of En expression. (K–K″) USP8 expression patterns in wing and leg discs (arrow and arrowhead, respectively) were determined by FISH with a DIG-labeled mRNA probe against USP8. Rhodamin (red) was used to detect the DIG-labeled mRNA, and DAPI (green, was false colored in green to provide better contrast) was used to label the nuclei. All wing imaginal discs shown in this study were oriented with anterior on the left and ventral on the top. (L) A wild-type adult wing showing interveins 1–5. (M–N) Wings from either male or female flies expressing Flag-Smo by MS1096 Gal4. The arrow in (M) indicates the overgrowth structures between Vein 2 and 3, and the arrow in (N) indicates the thickened Vein 3 caused by the overexpression of Smo. (O) A wing from flies expressing USP8RNAi by MS1096 Gal4. (P–Q) Wings from male or female flies expressing Flag-USP8 by MS1096 Gal4. Arrows indicate the wing overgrowth structure or the thickness of Vein 3. Of note, males of the same genotype have more severe phenotypes than females, presumably due to dosage compensation of the X-chromosome carrying the MS1096-Gal4. (R) A wing from ptc mutant flies indicates the overgrowth between Vein 2 and 3. (S–T) Adult wings from flies expressing Flag-USP8 by MS1096 Gal4 in the background of ptc mutants. In the ptc heterozygous or homozygous background, USP8 induced more severe wing overgrowth, which is indicated by wing blisters (S) or sick wings (T).
Figure 7
Figure 7. USP8 promotes Smo signaling activity.
(A–B) Wild-type wing discs were stained with anti-β-Gal and anti-Ptc antibodies to show dpp-lacZ and endogenous Ptc expression, respectively. (C–L) Wing discs expressing GFP-Smo alone (C–D), Flag-USP8 alone (E–F), GFP-Smo and Flag-USP8 together (G–H), HA-USP8NT3 alone (I–J), or GFP-Smo and HA-USP8NT3 together (K–L) by MS1096 Gal4 were stained for dpp-LacZ or Ptc. The red arrows indicate the ectopic expression of either dpp-lacZ or Ptc. (M–M′) A wild-type wing disc was immunostained for ptc-lacZ and En. (N–O′) Wing discs expressing SmoSD123 alone or together with USP8RNAi by MS1096 Gal4 was immunostained for ptc-lacZ and En. Knockdown of USP8 by RNAi attenuated the ectopic ptc-lacZ expression (arrow in O) and greatly reduced the ectopic en expression (arrow in O′).
Figure 8
Figure 8. The deubiquitinase activity of USP8 variants.
(A) A diagram of the USP8 protein and deletion constructs. The interaction between Smo and USP8 was determined by immunoprecipitation experiments shown in Figure S2. The activity of each USP8 construct was assessed in S2 cells by their ability to regulate the levels of Smo ubiquitination (shown below). MIT, microtubule interacting and transport domain; RHOD, Rhodanese homology domain. (B) S2 cells were transfected with Myc-Smo and the indicated USP8 constructs followed by the immunoprecipitation assay to detect Smo ubiquitination. The expression of USP8 variants was detected by a Western blot of lysates with an anti-Flag antibody (lower-middle panel). The arrow indicates a non-specific band and the asterisks indicate a possible modification of USP8C>S or USP8NT3C>S. USP8NT3C>S and USP8CT1 did not regulate Smo ubiquitination (lanes 5 and 6, top panel). (C) The effects of USP8 mutants on Smo ubiquitination were examined in S2 cells by the immunoprecipitation assay. The asterisks indicate a possible modification of USP8. Of note, both USP8C>S and USP8NT1C>S have dominant negative effects on Smo ubiquitination (lanes 3 and 4, top panel), whereas USP8NT2C>S and USP8NT3C>S do not (lanes 5 and 6, top panel). (D–E′) Wing discs expressing USP8NT3 or USP8NT3C>S by ap-Gal4 was immunostained for Smo and HA. The arrow and arrowhead in (D) indicate elevated Smo accumulation in A- and P-compartment cells. Notably, expression of USP8NT3C>S has no effect on Smo accumulation.
Figure 9
Figure 9. The localization of USP8 variants in S2 cells.
S2 cells were cotransfected with USP8 constructs and RFP-Rab5 followed by immunostaining with an anti-HA antibody to label the expression of USP8 constructs. Rab5 marks the early endosome. Of note, the expression of USP8C>S and USP8NT1C>S causes enlarged early endosomes, and both forms of USP8 localize in these enlarged early endosomes. USP8, USP8NT1, USP8NT2, and USP8NT2C>S are evenly distributed in the cytosol without a specific pattern. USP8NT3 behaves like USP8NT2 and USP8NT3C>S behaves like USP8NT2C>S (unpublished data).
Figure 10
Figure 10. USP8 is required for Hh-induced Smo cell surface accumulation.
(A–B′) Wing discs expressing UAS-ShiRNAi or UAS- Shi-DNS (a strong line) by MS1096 Gal4 was stained for Smo and Ci. The arrows indicate the RNAi-mediated increase in accumulation of Smo and Ci. (C–O) CFP-Smo or CFP-SmoSD123 was transfected into S2 cells and the cells were treated with GFP dsRNA, Shi dsRNA, USP8 dsRNA, or Hh-conditioned medium. Smo that localized to the cell surface was visualized by immunostaining with an anti-SmoN antibody before membrane permeabilization. The total amount of expressed Smo was indicated by the CFP signal. Representative images are shown here. (P) Quantification analysis of the percentage of Smo on the cell surface (mean ± s.d.; n≥15). Ratio (%) = (cell surface signal/whole cell signal)×100.
Figure 11
Figure 11. USP8 prevents Smo localization in the early endosome.
(A) Illustration of the antibody uptake experiment. The red arrowheads indicate the route of the endocytosed anti-SmoN antibody. (B–F) S2 cells were transfected with CFP-Smo and treated with (B) control medium, (C) Hh-conditioned medium, (D) Shi dsRNA, (E) USP8 dsRNA, or (F) co-transfected with HA-USP8. The images shown here are from intensity projection over the z-axis. Hh largely inhibited the endocytosis of Smo, as indicated by the co-localization of Rab5 and SmoN (C). Reduction of Shi by RNAi and overexpression of USP8 have similar effects in the prevention of Smo localization in the early endosomes that are labeled by Rab5 staining (D and F). Knockdown of USP8 by RNAi caused the accumulation of Smo in enlarged early endosomes (E). Rab5 positive endosomes were identified based on Z-series confocal images of S2 cells stained with anti-Rab5 antibody. (G) The percentage of Smo that co-localized with Rab5 (SmoN staining) compared to the total amount of Smo (CFP signal) calculated from (B–F) (n≥10). (H) A model for Smo trafficking. USP8 is required for the Hh-induced cell surface accumulation of Smo. Hh blocks Shi-mediated Smo endocytosis. USP8 prevents Smo localization in the early endosome by deubiquitinating Smo, and promotes Smo phosphorylation and activation.
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References

    1. Beachy P. A, Karhadkar S. S, Berman D. M. Tissue repair and stem cell renewal in carcinogenesis. Nature. 2004;432:324–331. - PubMed
    1. Ingham P. W, McMahon A. P. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001;15:3059–3087. - PubMed
    1. Jiang J, Hui C. C. Hedgehog signaling in development and cancer. Dev Cell. 2008;15:801–812. - PMC - PubMed
    1. Zheng X, Mann R. K, Sever N, Beachy P. A. Genetic and biochemical definition of the Hedgehog receptor. Genes Dev. 2010;24:57–71. - PMC - PubMed
    1. Jia J, Jiang J. Decoding the Hedgehog signal in animal development. Cell Mol Life Sci. 2006;63:1249–1265. - PMC - PubMed

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